Project Summary In this proposal, we combine two powerful nanotechnologies, i.e., nanobodies and nanoscale scanning electrochemical microscopy (SECM), to gain an unprecedented understanding of molecular transport through the nuclear pore complex (NPC) as the sole gate between the cytoplasm and nucleus of a eukaryotic cell. We engineer nanobodies from camelid-derived heavy-chain antibodies against distinct components of NPC, i.e., nucleoporins, to innovatively examine our hypotheses that are significant fundamentally in biology and practically in biomedicine to synergistically advance human health care. The proposed work pioneers the application of anti-nucleoporin nanobodies as selective blockers of NPC to reveal the unique role of each nucleoporin in the regulation of nucleocytoplasmic molecular transport. We apply nanobodies to test our original hypothesis that nucleoporins are heterogeneously distributed through the NPC nanopore to constitute central and peripheral pathways. We employ nanoscale SECM developed in our laboratory to spatially resolve nanobody-blocked and unblocked pathways based on low and high passive permeability to small probe ions, respectively, thereby locating each nucleoporin within the nanopore. Furthermore, we apply nanobodies to assess our new hypothesis that nucleoporins possess various populations of hydrophobic and charged amino acids to sort out different macromolecules into different pathways not exclusively by hydrophobic interactions as a long-standing consensus, but cooperatively with electrostatic interactions. We challenge the consensus by investigating the passive transport of neurotoxic polydipeptides based on hydrophobic proline and cationic arginine, which were recently found to block the NPC as a potential common cause of genetic neurodegenerative diseases. We employ SECM to determine the high permeability of NPC to a proline? arginine polydipeptide and its analogs with various hydrophobicity or charges. The measured permeability will be affected differently by nanobodies that bind nucleoporins complimentarily or competitively with polydipeptides. Accordingly, this study will provide the identity of nucleoporins targeted by polydipeptides in addition to the type and strength of polydipeptide?nucleoporin interactions. These SECM studies of passive transport lay the foundation for fluorescence transport studies of passively impermeable macromolecules, which can be chaperoned through the NPC by nuclear transport receptors, i.e., importins, as a crucial step to gene expression regulation and gene delivery. We employ nanobodies to determine whether importins chaperon macromolecules through the peripheral pathway by utilizing both hydrophobic and anionic binding sites to recognize peripheral nucleoporins with hydrophobic and cationic amino acids. In addition, we assess whether the neurotoxicity of polydipeptides is related to their capability to block importin-facilitated macromolecular transport. Overall, the proposed work will provide fundamentally novel chemical insights to advance the rational design of genetic therapeutics for efficient and safe nuclear import through the NPC.